The present invention relates generally to the fields of genes and promoters. More particularly, the present invention relates to an isolated gene and promoter of the enzyme ethanolaminephosphate cytidylyltransferase.
Phosphatidylethanolamine (PE) is an abundant lipid in both eukaryotic and prokaryotic cells. PE is situated primarily on the inner leaflet of the cell membrane where it interacts with inner-membrane proteins (1) or acts as a molecular chaperone and assists in proper protein folding (2). PE also plays an important role in physiological processes such as blood coagulation, platelet activation, cell signalling, membrane fusion, cell cycle progression, cell division, and apoptosis (3-9). Transfer of PE from lipoproteins to platelets induces their activation (3) and PE induces high-affinity binding sites for factor VIII and stimulates its pro-coagulant activity (4). PE is also involved in the thrombotic activity found in some cases of lupus where it further inhibits activated protein C (5). PE is a direct precursor of other lipids and provides ethanolamine moiety for anandamide (a physiological ligand for the cannabioid receptors) and glycosylphospatidylinositol (GPI) membrane anchors for a diverse group of proteins known as proteoglycans.
Distribution of PE in membranes plays a pivotal role during cytokinesis (7). Prior to late telophase, PE becomes exposed on the cell surface at the cleavage furrow, where it regulates the movement of the actin contractile ring and plasma membrane (7). Interestingly, the surface trapping of PE causes cell arrest (10), and the appearance of PE (together with PS) on the cell surface is an early hallmark of apoptosis (8). Products of PE metabolism, fatty acids, diacylglycerols and phosphatidic acid serve a critical role as second messengers in various signalling pathways and PE is an immediate donor of phosphoethanolamine residue linking glycosylphosphatydylinositol (GPI) anchor to proteins (11-14). There exist specialized forms of PE such as plasmalogens and derivatives such as natural cannabinoid anandamide and glycosylated PE (15-17). Plasmalogens play a role in the prevention of oxidation of lipoproteins (16) and constitute a significant portion of total PE in many tissues (18,19). However, despite their relative abundance, the principal biological function of plasmalogens is not firmly established and the understanding of the regulation of their production is surprisingly limited. Their production is severely impaired in the peroxisomal disorders such as Zellweger syndrome, Refsum disease (11) and neurological disorders (21-23). Glycosylated PE is abundant in lipoproteins of diabetics and has been implicated in the promotion of atherosclerosis in those individuals (24).
There are several pathways for the biosynthesis of PE, certain of which form PE from the alteration of other lipids. These include the decarboxylation of phosphatidylserine (PS) by a PS decarboxylase (PSD) and the base-exchange reaction with PS by a PS synthase (PSS) or phosphatydylcholine (PC). The third pathway, the CDP-ethanolamine pathway or Kenedy pathway, synthesizes PE de novo from ethanolamine and diacylglycerols (DAGs). The CDP-ethanolamine pathway includes three enzymatic steps consisting of the phosphorylation of ethanolamine (Etn), the formation of CDP-ethanolamine and pyrophosphate from phosphoethanolamine (P-Etn), and the final formation of PE from the transfer of phosphoethanolamine from CDP-ethanolamine (CDP-Etn) to diacylglycerol (DAG). These three steps are catalyzed by the enzymes ethanolamine kinase (EK), CTP:phosphoethanolamine cytidylyltransferase (ET), and ethanolaminephosphotransferase (EPT), respectively as shown in FIG. 1.
Little is known about genomic regulation of the biosynthesis of phospholipids. Several control points for the regulation of PE biosynthesis have been suggested. The reaction catalyzed by CTP: ethanolaminephosphate cytidilyltransferase (ET) has been suggested as a major regulatory step in the PE biosynthetic pathway. Considerable effort has been focused on the regulation of genes that encode enzymes in the fatty acid and cholesterol synthesis pathways. Promoters of these genes contain sterol-regulatory elements and are regulated by cholesterol-responsive transcription factors, sterol regulatory element binding proteins (SREBPs). However, lipogenic enzymes are mainly regulated by dietary carbohydrates, and their promoters contain insulin-response elements. The role for SREBPs in the regulation of fatty acid genes has been ascribed as means for cholesterol regulation of membrane phospholipids, typified in phosphatydyl choline production but direct regulation with cholesterol has also been suggested.
Studies on the regulation of genes that encode phospholipid biosynthetic enzymes have lagged behind that of other classes of lipogenic genes, primarily because most phospholipid-biosynthetic enzymes are difficult to isolate owing to their association with membranes. No evidence for direct transcriptional control of phospholipid genes with cholesterol, fatty acids or carbohydrates has yet been produced, but it is possible that these factors may have influence. Future experimentation with a combination of different transgenic models may determine specific genetic links for carbohydrate, cholesterol and phospholipid metabolism. Furthermore, additional links with regulators of lipid metabolism, including the peroxisome proliferator activated receptors (PPARs) and lipoproteins may be found. PPARs are activated by a diverse group of pharmacological ligands, the peroxisome proliferators (e.g., fibrates, troglitazone), which are well known drugs for regulating lipoprotein levels and very important for prevention of atherosclerosis.
Ethanolamine kinase (EK) exists in several isoforms (20,25,26) having both EK and choline kinase (CK) activities. The isolation of two rat cDNA clones for CK/EK has allowed for the characterization of two separate rat genes (27, 28) and two mouse gene products (29). Unlike CK/EK, EPT is responsible for production of PE by transferring phosphoethanolamine from CDP-ethanolamine to DAG (30) and a separate enzyme, cholinephophotransferase (CPT), is responsible for this reaction in the CDP-choline pathway. EPT and CPT are encoded by two separate genes (31,32). The EPT gene was cloned by complementation of an EPT yeast mutant with a yeast genomic library (33). Subsequently, the human cDNA for EPT has been isolated (34). Interestingly, the human EPT protein has broad substrate specificity, and has the ability to form both choline and ethanolamine lipids (34).
CTP: phosphoethanolamine cytidylyltransferase (ET) is one of the most substrate-specific and the most regulatory enzyme in the CDP-ethanolamine pathway (35). Only rat ET protein has been successfully purified and its biochemical properties clearly established (36-38). The rat protein is considered soluble but could localize between the cisternae of the rough ER and the cytosolic space suggesting some associations with membranes (37). Unlike CK/EK and EPT, rat ET only has activity towards ethanolaminephosphate and does not show any affinity for cholinephosphate (38). These findings strongly agree with genetic evidence indicating that ET and CTP: phosphocholine cytidylyltransferase (CT) cDNAs are produced by two different genes (39, 40). ET cDNAs from yeast, human, and rat have been functionally characterized and showed a high degree of homology between sequences (39-41). Neither the mouse ET cDNA nor any ET gene has yet been characterized.
An EST (GenBank(trademark) Accession No. BC003473) encoding 1855 bp of mRNA for the full-length mouse ET was identified, and is highly homologous to rat and human cDNAs, particularly in the proximity of the translation start codon ATG as shown in FIG. 2. Computer analysis suggests that ET protein possesses a recognition motif MIRNG and two catalytic domains with large internal repetitive sequences in its N-and C-terminal halves; both parts of the sequence contain the CTP-binding motif HXGH (41), which is conserved in the entire cytidylyltransferase superfamily (42). CT does not contain the MIRNG motif, does not have two similar halves, and possesses only single HXGH motif (39, 40).
Even though ET is a critical enzyme required for the de novo synthesis of PE, no gene has been characterized and little is known about the regulation of this enzyme""s expression. Recent evidence (43) indicates that rat ET mRNA and protein level increase during liver development; a higher change in mRNA than protein was visible, suggesting that combined transcriptional and translational events are may be involved in the regulation of ET activity. It would be advantageous to fully elucidate the regulation of the ET gene product.
Little effort has been given towards the study of ET regulation at the genetic level. This is due to the fact that it was often assumed that ET is similar to CTP: phosphocholine cytidylyltransferase (CT), the major regulatory enzyme in the CDP-choline pathway for the biosynthesis of phosphatidylcholine (PC) (44-48). It was not until recently that it was speculated that ET was regulated in a different manner than CT (49) and that PE""s importance may not lie in the fact that it only resides in cell membranes but rather that it can be found in other forms that could play vital roles in proper cell functioning. Further examples of this are PE plasmalogens, which comprise a large portion of total cellular PE (50) and may play an important role in proper functioning of the brain and heart (51). It would be of great interest to fully understand the regulation of ET at the level of transcription. This information could be compared to the regulatory pathways of CT and other genes involved in lipid and cholesterol metabolism to establish the regulatory mechanisms for membrane biogenesis and lipid maintenance during normal development and in lipid-related pathological states.
It is an object of the present invention to provide a nucleotide sequence of a murine ET gene. It is yet another object of the present invention to provide a promoter sequence of the human ET gene.
The invention provides a gene encoding a protein having ethanolaminephosphate cytidylyltransferase activity consisting of a sequence selected from the group consisting of: (a) SEQ ID NO:1; (b) a degenerate sequence of SEQ ID NO:1 and (c) a sequence which hybridizes to the complement of SEQ ID NO:1 under stringent conditions.
Further, the invention provides a promoter of an ethanolaminephosphate cytidylyltransferase gene, said promoter consisting of a sequence selected from the group consisting of: (a) SEQ ID NO:2; (b) a sequence according to SEQ ID NO:2 having substitutions or deletions and maintaining promoter activity; and (c) a sequence which hybridizes to the complement of SEQ ID NO:2 under stringent conditions.
The gene and promoter according to the invention can be used to produce a transgenic mammal. The gene and promoter are useful in identifying, preventing, and treating diseases related to inappropriate phoshatidylethanolamine production.
Other aspects and features of the present invention will become apparent to those ordinarily skilled in the art upon review of the following description of specific embodiments of the invention in conjunction with the accompanying figures.